Compositions and methods for inhibiting expression of factor vii gene

ABSTRACT

The invention relates to a double-stranded ribonucleic acid (dsRNA) for inhibiting the expression of the Factor VII gene.

RELATED APPLICATIONS

This is a continuation of U.S. application Ser. No. 14/156,193, filedJan. 15, 2014, which is a continuation of U.S. application Ser. No.13/619,657, filed on Sep. 14, 2012, now U.S. Pat. No. 8,664,193, whichis a continuation of U.S. application Ser. No. 12/970,673, filed Dec.16, 2010, now U.S. Pat. No. 8,334,273, which is a continuation of U.S.application Ser. No. 12/331,708, filed on Dec. 10, 2008, now U.S. Pat.No. 7,871,985, which claims benefit of U.S. Provisional Application No.61/012,670, filed Dec. 10, 2007, and U.S. Provisional Application No.61/014,879, filed Dec. 19, 2007, which are incorporated herein in theirentirety by reference, for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OF DEVELOPMENT

This invention was made with government support under Grant Nos.HHSN26620060012C awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to double-stranded ribonucleic acid (dsRNA), andits use in mediating RNA interference to inhibit the expression of theFactor VII gene and the use of the dsRNA to treat or prevent a FactorVII-mediated disorder, e.g., Viral Hemorrhagic Fever.

BACKGROUND OF THE INVENTION

Factor VII (FVII) is involved in coagulation. Upon blood vessel injury,tissue factor (TF), located on the outside of vessels, is exposed to theblood and circulating factor VII. Once bound to TF, FVII is activated toFVIIa by various proteases, including thrombin (factor IIa), activatedfactor X and the FVIIa-TF complex itself. In addition to its role ininitiating coagulation, the TF/FVIIa complex has been reported to havedirect proinflammatory effects independent of the activation ofcoagulation.

A number of viruses have been reported to cause lethal hemorrhagicdisease in humans and certain other primates. These viruses are from anumber of viral families including Filoviridae, Arenaviridae,Bunyaviridae, and Flaviridae. Patients affected with hemorrhagic feverstypically develop a severe consumptive disseminated intravascularcoagulation (DIC). DIC is characterized by wide-spread systematicactivation of the coagulation cascade resulting in excess thrombingeneration. In addition, activation of the fibrinolytic system coupledwith the consumption of coagulation factors results in a depletion ofclotting factors and degradation of platelet membrane glycoproteins.

Certain infectious agents are also known to activate the coagulationsystem following infection. A variety of inflammatory stimuli, includingbacterial cell products, viral infection and cytokines have beenreported to induce the expression of TF on the surface of endothelialcells and monocytes, thereby activating the coagulation pathway.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). WO 99/32619 (Fire et al.) discloses the use of adsRNA of at least 25 nucleotides in length to inhibit the expression ofthe unc-22 gene in C. elegans. dsRNA has also been shown to degradetarget RNA in other organisms, including plants (see, e.g., WO 99/53050,Waterhouse et al.; and WO 99/61631, Heifetz et al.), Drosophila (see,e.g., Yang, D., et al., Curr. Biol. (2000) 10:1191-1200), and mammals(see WO 00/44895, Limmer; and DE 101 00 586.5, Kreutzer et al.).

SUMMARY OF THE INVENTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the FactorVII gene in a cell or mammal using such dsRNA. The invention alsoprovides compositions and methods for treating pathological conditionsand diseases caused by expression of the Factor VII gene, such ascoagulation disorders, including viral hemorrhagic fever. The dsRNAfeatured in the invention includes an RNA strand (the antisense strand)having a region that is less than 30 nucleotides in length, generally19-24 nucleotides in length, and which is substantially complementary orfully complementary to the corresponding region of an mRNA transcript ofthe Factor VII gene.

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of the Factor VIIgene. The dsRNA includes at least two sequences that are complementary,e.g., substantially complementary, fully complementary, or sufficientlycomplementary to hybridize under physiological conditions, to eachother. The dsRNA includes a sense strand including a first sequence andan antisense strand including a second sequence. The antisense strandincludes a nucleotide sequence which is substantially or fullycomplementary to the corresponding region of an mRNA encoding FactorVII, and the region of complementarity is less than 30 nucleotides inlength, generally 19-24 nucleotides, e.g., 19 to 21 nucleotides inlength. In some embodiments, the dsRNA is from about 10 to about 15nucleotides, and in other embodiments the dsRNA is from about 25 toabout 30 nucleotides in length. In one embodiment the dsRNA, uponcontacting with a cell expressing the Factor VII, inhibits theexpression of the Factor VII gene by at least 25%, e.g., by at least35%, or by at least 40%. In one embodiment, the Factor VII dsRNA isformulated in a stable nucleic acid particle (SNALP).

In one embodiment, the dsRNA can reduce mRNA levels by at least 40%,60%, 80%, or 90%, e.g., as measured by an assay described herein. Forexample, the dsRNA can reduce liver Factor VII mRNA levels in rats by atleast 40%, 60%, 80%, or 90%, such as with a single administration of adose of 98N12-5 formulated Factor VII-targeting siRNA. In anotherembodiment, the dsRNA produces similar reduction in protein levels,e.g., as measured by an assay described herein. In yet anotherembodiment, a single injection of a 98N12-5 formulated FactorVII-targeting siRNA (siFVII) can mediate silencing for 1, 2, 3 or 4weeks or more, e.g., as measured by an assay described herein. Assays tomeasure FVII mRNA and protein levels can also be performed by standardmethods known in the art. For example, FVII mRNA can be measured byRT-PCR or Northern blot analysis. FVII protein levels can be measured byenzymatic assay, or by antibody-based methods, e.g., Western blot,ELISA, or immunohisto chemistry.

The dsRNA molecules targeting FVII can include a first sequence of thedsRNA that is selected from the group consisting of the sense sequencesof Tables 1, 2, and 3, and the second sequence is selected from thegroup consisting of the antisense sequences of Tables 1, 2, and 3. ThedsRNA molecules featured in the invention can include naturallyoccurring nucleotides or can include at least one modified nucleotide,such as a 2′-O-methyl modified nucleotide, a nucleotide including a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or dodecanoic acid bisdecylamide group.Alternatively, the modified nucleotide may be chosen from the group of:a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modifiednucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.Generally, the first sequence of said dsRNA is selected from the groupconsisting of the sense sequences of Tables 1, 2, and 3, and the secondsequence is selected from the group consisting of the antisensesequences of Tables 1, 2, and 3.

In another embodiment, the invention provides a cell including dsRNAtargeting FVII. The cell is generally a mammalian cell, such as a humancell.

In another embodiment, the invention provides a pharmaceuticalcomposition for inhibiting the expression of the Factor VII gene in anorganism, including one or more of the dsRNA targeting FVII, and apharmaceutically acceptable carrier.

In another embodiment, the invention provides a method for inhibitingthe expression of the Factor VII gene in a cell, including the followingsteps:

-   -   (a) introducing into the cell a double-stranded ribonucleic acid        (dsRNA), wherein the dsRNA includes at least two sequences that        are complementary, e.g., substantially or fully complementary,        to each other; and    -   (b) maintaining the cell produced in step (a) for a time        sufficient to obtain degradation of the mRNA transcript of the        Factor VII gene, thereby inhibiting expression of the Factor VII        gene in the cell.

The dsRNA includes a sense strand including a first sequence and anantisense strand including a second sequence. The antisense strandincludes a region of complementarity which is substantially or fullycomplementary to the corresponding region of an mRNA encoding FactorVII, and where the region of complementarity is less than 30 nucleotidesin length, generally 19-24 nucleotides in length, and where the dsRNA,upon contact with a cell expressing Factor VII, inhibits expression ofthe Factor VII gene by at least at least 40%. In one embodiment, thedsRNA can reduce mRNA by at least 40%, 60%, 80%, or 90%, e.g., asmeasured by an assay described herein. For example, the dsRNA can reduceliver Factor VII mRNA levels in rats by at least 40%, 60%, 80%, or 90%following a single administration of a dose of 98N12-5 formulated FactorVII-targeting siRNA. In one embodiment the dsRNA produce similarreductions in protein levels, e.g., as measured by an assay describedherein. In another embodiment, a single injection of 98N12-5 formulatedFactor VII-targeting siRNA (siFVII) can mediate silencing for 1, 2, 3 or4 weeks or more, e.g., as measured by an assay described herein.

In another embodiment, the invention provides methods for treating,preventing or managing a Factor VII-mediated disorder by administeringto a patient in need of such treatment, prevention or management atherapeutically or prophylactically effective amount of one or more ofthe dsRNAs featured in the invention.

In one embodiment, a FVII dsRNA can be used to treat a hemorrhagicfever, such as a viral hemorrhagic fever. Such a fever can be cause by avirus, such as a virus from the Filoviridae, Arenaviridae, Bunyaviridae,or Flaviridae families. For example, a FVII dsRNA can used to treat ahemorrhagic fever caused be a virus from the Filoviridae family, e.g.,an Ebola or Marburg virus, or a virus from the Arenaviridae family,e.g., a Lassa virus.

In another embodiment, a FVII dsRNA featured herein is used to treat acoagulopathy or an inflammatory response, such as may be caused by ahemorrhagic fever.

In another embodiment, a FVII dsRNA can be used to treat a thromboticdisorder, e.g., a local thrombus, such as may arise from the rupture ofatherosclerotic plaque. In another embodiment, administration of a FVIIdsRNA is used to treat or prevent acute myocardial infarction orunstable angina. A FVII dsRNA can also be used to treat an occlusivecoronary thrombus. In another embodiment, a FVII dsRNA is administeredto treat or prevent deep vein thrombosis. In yet another embodiment, aFVII dsRNA is administered to treat or prevent a venous thromboembolism,e.g., in a cancer patients.

In another embodiment, a FVII dsRNA is administered to a patient, andafter 1, 2, 3, or 4 weeks, the patient is tested to determine FVII mRNAlevels, e.g., in the blood or urine, or in a particular tissue, e.g.,the liver. If the level of FVII mRNA is determined to be above a pre-setlevel, the patient will be administered another dose of FVII dsRNA. Ifthe level of FVII mRNA is determined to be below the pre-set level, thepatient is not administered another dose of the FVII dsRNA. In yetanother embodiment, a FVII dsRNA is administered to treat aproliferative disorder, e.g., cancer, such as ovarian, breast, head andneck, prostate, colorectal or lung cancer.

It has been discovered that a single administration can provideprolonged silencing. Thus, in another embodiment, a dose of FVII dsRNAis administered to a patient and the dose is sufficient that Factor VIImRNA or protein is: less than or equal to 20% of pretreatment levels (orthe levels which would be seen in the absence of treatment) for at least5, 10, or 15 days post-treatment; less than or equal to 40% ofpretreatment levels (or the levels which would be seen in the absence oftreatment) for at least 5, 10, or 15 days post-treatment; less than orequal to 60% of pretreatment levels (or the levels which would be seenin the absence of treatment) for at least 5, 10, 15, or 20 dayspost-treatment; less than or equal to 80% of pretreatment levels (or thelevels which would be seen in the absence of treatment) for at least 5,10, 15, 20, or 25 days post-treatment.

In one embodiment, a dose is administered and no additional dose of FVIIdsRNA is administered for at least 5, 10, 15, 20, or 25 days after thefirst administration or course of administrations is finished.

In another embodiment, the invention provides vectors for inhibiting theexpression of the Factor VII gene in a cell, including a regulatorysequence operably linked to a nucleotide sequence that encodes at leastone strand of one of the dsRNA featured in the invention.

In another embodiment, the invention provides a cell including a vectorfor inhibiting the expression of the Factor VII gene in a cell. Thevector includes a regulatory sequence operably linked to a nucleotidesequence that encodes at least one strand of one of the dsRNA featuredin the invention.

TABLE 1 FVII dsRNAs (modified). Sense antisense strand strand SEQ SEQ IDID duplex name NO: sequence (5′-3′) name NO: sequence (5′-3′) nameA26884  5 GAcGcuGGccuucGuGcGcdTsdT A26885  6 GCGcACGAAGGCcAGCGUCdTsdTAD16734 A26886  7 ccucuGccuGcccGAAcGGdTsdT A26887  8CCGuuCGGGcAGGcAGAGGdTsdT AD16735 A26888  9 ccuucGAGGGccGGAAcuGdTsdTA26889 10 cAGuuCCGGCCCUCGAAGGdTsdT AD16736 A26890 11ccAAccAcGAcAucGcGcudTsdT A26891 12 AGCGCGAuGUCGuGGuuGGdTsdT AD16737A26892 13 cucccAGuAcAucGAGuGGdTsdT A26893 14 CcACUCGAuGuACuGGGAGdTsdTAD16738 A26894 15 cAAccAcGAcAucGcGcuGdTsdT A26895 16cAGCGCGAuGUCGuGGuuGdTsdT AD16739 A26896 17 cAGuccuAuAucuGcuucudTsdTA26897 18 AGAAGcAGAuAuAGGACuGdTsdT AD16740 A26898 19ccAuGGcAGGuccuGuuGudTsdT A26899 20 AcAAcAGGACCuGCcAuGGdTsdT AD16741A26900 21 cucuGccuGcccGAAcGGAdTsdT A26901 22 UCCGuuCGGGcAGGcAGAGdTsdTAD16742 A26902 23 cGGcGGcuGuGAGcAGuAcdTsdT A26903 24GuACuGCUcAcAGCCGCCGdTsdT AD16743 A26904 25 uucuGuGccGGcuAcucGGdTsdTA26905 26 CCGAGuAGCCGGcAcAGAAdTsdT AD16744 A26906 27GAccAGcuccAGuccuAuAdTsdT A26907 28 uAuAGGACuGGAGCuGGUCdTsdT AD16745A26908 29 uuGuuGGuGAAuGGAGcucdTsdT A26909 30 GAGCUCcAuucACcAAcAAdTsdTAD16746 A26910 31 AuGuGGAAAAAuAccuAuudTsdT A26911 32AAuAGGuAuuuuUCcAcAUdTsdT AD16747 A26912 33 GuGGuccucAcuGAccAuGdTsdTA26913 34 cAuGGUcAGuGAGGACcACdTsdT AD16748 A26914 35AcGAcAucGcGcuGcuccGdTsdT A26915 36 CGGAGcAGCGCGAuGUCGUdTsdT AD16749A26916 37 cAAGGAccAGcuccAGuccdTsdT A26917 38 GGACuGGAGCuGGUCCuuGdTsdTAD16750 A26918 39 GcAAGGAccAGcuccAGucdTsdT A26919 40GACuGGAGCuGGUCCuuGCdTsdT AD16751 A26920 41 AAGGAccAGcuccAGuccudTsdTA26921 42 AGGACuGGAGCuGGUCCuudTsdT AD16752 A26922 43ccAGGGucucccAGuAcAudTsdT A26923 44 AuGuACuGGGAGACCCuGGdTsdT AD16753A26924 45 cAuGGcAGGuccuGuuGuudTsdT A26925 46 AAcAAcAGGACCuGCcAuGdTsdTAD16754 A26926 47 AcGGcGGcuGuGAGcAGuAdTsdT A26927 48uACuGCUcAcAGCCGCCGUdTsdT AD16755 A26928 49 cuGuGAGcAGuAcuGcAGudTsdTA26929 50 ACuGcAGuACuGCUcAcAGdTsdT AD16756 A26930 51cGGuGcuGGGcGAGcAcGAdTsdT A26931 52 UCGuGCUCGCCcAGcACCGdTsdT AD16757 “s”indicates a phosphorothioate linkage; 2′-O-Me modified nucleotides areindicated by lower case.

TABLE 2 FVII dsRNAs (unmodified). SEQ sense ID strand NO:sequence (5′-3′) 53 GACGCUGGCCUUCGUGCGC 55 CCUCUGCCUGCCCGAACGG 57CCUUCGAGGGCCGGAACUG 59 CCAACCACGACAUCGCGCU 61 CUCCCAGUACAUCGAGUGG 63CAACCACGACAUCGCGCUG 65 CAGUCCUAUAUCUGCUUCU 67 CCAUGGCAGGUCCUGUUGU 69CUCUGCCUGCCCGAACGGA 71 CGGCGGCUGUGAGCAGUAC 73 UUCUGUGCCGGCUACUCGG 75GACCAGCUCCAGUCCUAUA 77 UUGUUGGUGAAUGGAGCUC 79 AUGUGGAAAAAUACCUAUU 81GUGGUCCUCACUGACCAUG 83 ACGACAUCGCGCUGCUCCG 85 CAAGGACCAGCUCCAGUCC 87GCAAGGACCAGCUCCAGUC 89 AAGGACCAGCUCCAGUCCU 91 CCAGGGUCUCCCAGUACAU 93CAUGGCAGGUCCUGUUGUU 95 ACGGCGGCUGUGAGCAGUA 97 CUGUGAGCAGUACUGCAGU 99CGGUGCUGGGCGAGCACGA SEQ antisense ID strand NO: sequence (5′-3′) 54GCGCACGAAGGCCAGCGUC 56 CCGUUCGGGCAGGCAGAGG 58 CAGUUCCGGCCCUCGAAGG 60AGCGCGAUGUCGUGGUUGG 62 CCACUCGAUGUACUGGGAG 64 CAGCGCGAUGUCGUGGUUG 66AGAAGCAGAUAUAGGACUG 68 ACAACAGGACCUGCCAUGG 70 UCCGUUCGGGCAGGCAGAG 72GUACUGCUCACAGCCGCCG 74 CCGAGUAGCCGGCACAGAA 76 UAUAGGACUGGAGCUGGUC 78GAGCUCCAUUCACCAACAA 80 AAUAGGUAUUUUUCCACAU 82 CAUGGUCAGUGAGGACCAC 84CGGAGCAGCGCGAUGUCGU 86 GGACUGGAGCUGGUCCUUG 88 GACUGGAGCUGGUCCUUGC 90AGGACUGGAGCUGGUCCUU 92 AUGUACUGGGAGACCCUGG 94 AACAACAGGACCUGCCAUG 96UACUGCUCACAGCCGCCGU 98 ACUGCAGUACUGCUCACAG 100 UCGUGCUCGCCCAGCACCG

TABLE 3 FVII dsRNAs (3′ dinucleotide modifications). SEQ ID sense strandNO: sequence (5′-3′) 101 GACGCUGGCCUUCGUGCGCNN 103 CCUCUGCCUGCCCGAACGGNN105 CCUUCGAGGGCCGGAACUGNN 107 CCAACCACGACAUCGCGCUNN 109CUCCCAGUACAUCGAGUGGNN 111 CAACCACGACAUCGCGCUGNN 113CAGUCCUAUAUCUGCUUCUNN 115 CCAUGGCAGGUCCUGUUGUNN 117CUCUGCCUGCCCGAACGGANN 119 CGGCGGCUGUGAGCAGUACNN 121UUCUGUGCCGGCUACUCGGNN 123 GACCAGCUCCAGUCCUAUANN 125UUGUUGGUGAAUGGAGCUCNN 127 AUGUGGAAAAAUACCUAUUNN 129GUGGUCCUCACUGACCAUGNN 131 ACGACAUCGCGCUGCUCCGNN 133CAAGGACCAGCUCCAGUCCNN 135 GCAAGGACCAGCUCCAGUCNN 137AAGGACCAGCUCCAGUCCUNN 139 CCAGGGUCUCCCAGUACAUNN 141CAUGGCAGGUCCUGUUGUUNN 143 ACGGCGGCUGUGAGCAGUANN 145CUGUGAGCAGUACUGCAGUNN 147 CGGUGCUGGGCGAGCACGANN SEQ antisense ID strandNO sequence (5′-3′ 102 GCGCACGAAGGCCAGCGUCNN 104 CCGUUCGGGCAGGCAGAGGNN106 CAGUUCCGGCCCUCGAAGGNN 108 AGCGCGAUGUCGUGGUUGGNN 110CCACUCGAUGUACUGGGAGNN 112 CAGCGCGAUGUCGUGGUUGNN 114AGAAGCAGAUAUAGGACUGNN 116 ACAACAGGACCUGCCAUGGNN 118UCCGUUCGGGCAGGCAGAGNN 120 GUACUGCUCACAGCCGCCGNN 122CCGAGUAGCCGGCACAGAANN 124 UAUAGGACUGGAGCUGGUCNN 126GAGCUCCAUUCACCAACAANN 128 AAUAGGUAUUUUUCCACAUNN 130CAUGGUCAGUGAGGACCACNN 132 CGGAGCAGCGCGAUGUCGUNN 134GGACUGGAGCUGGUCCUUGNN 136 GACUGGAGCUGGUCCUUGCNN 138AGGACUGGAGCUGGUCCUUNN 140 AUGUACUGGGAGACCCUGGNN 142AACAACAGGACCUGCCAUGNN 144 UACUGCUCACAGCCGCCGUNN 146ACUGCAGUACUGCUCACAGNN 148 UCGUGCUCGCCCAGCACCGNN N indicates anynucleotide (G, A, C, T)

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a bar graph showing liver FVII mRNA levels followingadministration of FVII siRNA.

FIG. 2 is a bar graph showing serum FVII protein levels followingadministration of FVII siRNA.

FIG. 3 is a bar graph showing prothrombin time following administrationof FVII siRNA.

FIG. 4 is a bar graph showing FVII protein levels in mouse hepatocytesfollowing treatment with a liposomally-formulated FVII dsRNA. Aliposomally-formulated luciferase dsRNA was used as a negative control.

FIG. 5 is a graph showing survival levels of mice infected with Ebola,and treated with FVII dsRNA. Negative controls included untreated miceand mice treated with a luciferase dsRNA.

FIG. 6 is a graph showing levels of Factor VII protein over time inC57BL/6 mice treated with a single bolus i.v. injection of LNP01-siFVIIat 5 mg/kg at timepoint 0.

FIGS. 7A and 7B represent the mRNA sequence (SEQ ID NO: 149) of thehuman FVII transcript variant at GenBank Accession Number NM_(—)000131.3(3141 bp) (GenBank record dated Nov. 18, 2007).

FIGS. 8A and 8B represent the mRNA sequence (SEQ ID NO: 150) of humanFVII transcript variant at GenBank Accession Number NM_(—)019616.2 (3141bp) (GenBank record dated Nov. 18, 2007).

FIGS. 9A and 9B represent the mRNA sequence (SEQ ID NO: 151) of rhesusFVII transcript variant at GenBank Accession Number NM_(—)001080136.1(2424 bp) (GenBank record dated Jan. 13, 2007).

FIG. 10 represents a partial cds sequence (SEQ ID NO: 152) of the Macacamulatta FVII at GenBank Accession Number D21212.1 (478 bp) (GenBankrecord dated Dec. 27, 2006).

FIGS. 11A, 11B, and 11C represent the sequence (SEQ ID NOS 153 and153-154, respectively, in order of appearance) of the Macaca mulattaFVII at ENSEMBLE accession no. EMSMMUT00000001477 (1389 bp).

FIG. 12 represents the sequence (SEQ ID NO: 155) of the Macaca mulattaFVII at ENSEMBLE accession no. EMSMMUT00000042997 (1326 bp).

DETAILED DESCRIPTION

The invention provides double-stranded ribonucleic acid (dsRNA), as wellas compositions and methods for inhibiting the expression of the FactorVII gene in a cell or mammal using the dsRNA. The invention alsoprovides compositions and methods for treating pathological conditionsand diseases in a mammal caused by the expression of the Factor VII geneusing dsRNA. dsRNA directs the sequence-specific degradation of mRNAthrough a process known as RNA interference (RNAi). The process occursin a wide variety of organisms, including mammals and other vertebrates.

The dsRNA featured in the invention includes an RNA strand (theantisense strand) having a region which is less than 30 nucleotides inlength, generally 19-24 nucleotides in length, and is substantially orfully complementary to at least part of an mRNA transcript of the FactorVII gene. The use of these dsRNAs enables the targeted degradation ofmRNAs of genes that are implicated in thrombosis in mammals. Usingcell-based and animal assays, the present inventors have demonstratedthat very low dosages of these dsRNA can specifically and efficientlymediate RNAi, resulting in significant inhibition of expression of theFactor VII gene. Thus, the methods and compositions featured in theinvention include dsRNAs useful for treating a thrombotic disorder.

The following detailed description discloses how to make and use thedsRNA and compositions containing dsRNA to inhibit the expression of atarget Factor VII gene, as well as compositions and methods for treatingdiseases and disorders caused by the expression of Factor VII, such as athrombotic disorder. The pharmaceutical compositions featured in theinvention include a dsRNA having an antisense strand having a region ofcomplementarity which is less than 30 nucleotides in length, generally19-24 nucleotides in length, and is substantially complementary to atleast part of an RNA transcript of the Factor VII gene, together with apharmaceutically acceptable carrier.

Accordingly, certain aspects of the invention provide pharmaceuticalcompositions including a dsRNA targeting FVII, together with apharmaceutically acceptable carrier, methods of using the compositionsto inhibit expression of the Factor VII gene, and methods of using thepharmaceutical compositions to treat diseases caused by expression ofthe Factor VII gene.

I. Definitions

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, and uracil as a base, respectively.However, it will be understood that the term “ribonucleotide” or“nucleotide” can also refer to a modified nucleotide, as furtherdetailed below, or a surrogate replacement moiety. The skilled person iswell aware that guanine, cytosine, adenine, and uracil may be replacedby other moieties without substantially altering the base pairingproperties of an oligonucleotide including a nucleotide bearing suchreplacement moiety. For example, without limitation, a nucleotideincluding inosine as its base may base pair with nucleotides containingadenine, cytosine, or uracil. Hence, nucleotides containing uracil,guanine, or adenine may be replaced in the nucleotide sequences featuredin the invention by a nucleotide containing, for example, inosine. Inanother example, adenine and cytosine anywhere in the oligonucleotidecan be replaced with guanine and uracil, respectively to form G-U Wobblebase pairing with the target mRNA. Sequences including such replacementmoieties are embodiments featured in the invention.

By “Factor VII” as used herein is meant a Factor VII mRNA, protein,peptide, or polypeptide. The term “Factor VII” is also known in the artas A132620, Cf7, Coagulation factor VII precursor, coagulation factorVII, FVII, Serum prothrombin conversion accelerator, FVII coagulationprotein, and eptacog alfa.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof the Factor VII gene, including mRNA that is a product of RNAprocessing of a primary transcription product.

As used herein, the term “strand including a sequence” refers to anoligonucleotide including a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used in the context of a nucleotide pair, means aclassic Watson-Crick pair, i.e., GC, AT, or AU. It also extends toclassic Watson-Crick pairings where one or both of the nuclotides hasbeen modified as decribed herein, e.g., by a rbose modification or aphosphate backpone modification. It can also include pairing with aninosine or other entity that does not substantially alter the basepairing properties.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide including the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide including thesecond nucleotide sequence, as will be understood by the skilled person.Complementarity can include, full complementarity, substantialcomplementarity, and sufficient complementarity to allow hybridizationunder physiological conditions, e.g, under physiologically relevantconditions as may be encountered inside an organism. Fullcomplementarity refers to complementarity, as defined above for anindividual pair, at all of the pairs of the first and second sequence.When a sequence is “substantially complementary” with respect to asecond sequence herein, the two sequences can be fully complementary, orthey may form one or more, but generally not more than 4, 3 or 2mismatched base pairs upon hybridization, while retaining the ability tohybridize under the conditions most relevant to their ultimateapplication. Substantial complementarity can also be defined ashybridization under stringent conditions, where stringent conditions mayinclude: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C.for 12-16 hours followed by washing. The skilled person will be able todetermine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA including one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide includes a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,may yet be referred to as “fully complementary” for the purposes of theinvention.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary”, “fully complementary”, “substantiallycomplementary” and sufficient complementarity to allow hybridizationunder physiological conditions, e.g, under physiologically relevantconditions as may be encountered inside an organism, may be usedhereinwith respect to the base matching between the sense strand and theantisense strand of a dsRNA, or between the antisense strand of a dsRNAand a target sequence, as will be understood from the context of theiruse.

As used herein, a polynucleotide which is “complementary, e.g.,substantially complementary to at least part of” a messenger RNA (mRNA)refers to a polynucleotide which is complementary, e.g., substantiallycomplementary, to a contiguous portion of the mRNA of interest (e.g.,encoding Factor VII). For example, a polynucleotide is complementary toat least a part of a Factor VII mRNA if the sequence is substantiallycomplementary to a non-interrupted portion of an mRNA encoding FactorVII.

The term “double-stranded RNA” or “dsRNA”, as used herein, refers to aribonucleic acid molecule, or complex of ribonucleic acid molecules,having a duplex structure including two anti-parallel and substantiallycomplementary, as defined above, nucleic acid strands. The two strandsforming the duplex structure may be different portions of one larger RNAmolecule, or they may be separate RNA molecules. Where the two strandsare part of one larger molecule, and therefore are connected by anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′end of the respective other strand forming the duplex structure,the connecting RNA chain is referred to as a “hairpin loop”. Where thetwo strands are connected covalently by means other than anuninterrupted chain of nucleotides between the 3′-end of one strand andthe 5′end of the respective other strand forming the duplex structure,the connecting structure is referred to as a “linker.” The RNA strandsmay have the same or a different number of nucleotides. The maximumnumber of base pairs is the number of nucleotides in the shortest strandof the dsRNA. In addition to the duplex structure, a dsRNA may compriseone or more nucleotide overhangs. A dsRNA as used herein is alsoreferred to as a “small inhibitory RNA,” “siRNA,” “iRNA agent” or “RNAiagent.”

As used herein, a “nucleotide overhang” refers to the unpairednucleotide or nucleotides that protrude from the duplex structure of adsRNA when a 3′-end of one strand of the dsRNA extends beyond the 5′-endof the other strand, or vice versa. “Blunt” or “blunt end” means thatthere are no unpaired nucleotides at that end of the dsRNA, i.e., nonucleotide overhang. A “blunt ended” dsRNA is a dsRNA that isdouble-stranded over its entire length, i.e., no nucleotide overhang ateither end of the molecule.

The term “antisense strand” refers to the strand of a dsRNA whichincludes a region that is substantially complementary to a targetsequence. As used herein, the term “region of complementarity” refers tothe region on the antisense strand that is substantially complementaryto a sequence, for example a target sequence, as defined herein. Wherethe region of complementarity is not fully complementary to the targetsequence, the mismatches are most tolerated in the terminal regions and,if present, are generally in a terminal region or regions, e.g., within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand,” as used herein, refers to the strand of a dsRNAthat includes a region that is substantially complementary to a regionof the antisense strand.

The term “identity” is the relationship between two or morepolynucleotide sequences, as determined by comparing the sequences.Identity also means the degree of sequence relatedness betweenpolynucleotide sequences, as determined by the match between strings ofsuch sequences. While there exist a number of methods to measureidentity between two polynucleotide sequences, the term is well known toskilled artisans (see, e.g., Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press (1987); and Sequence Analysis Primer,Gribskov., M. and Devereux, J., eds., M. Stockton Press, New York(1991)). “Substantially identical,” as used herein, means there is avery high degree of homology (e.g., 100% sequence identity) between thesense strand of the dsRNA and the corresponding part of the target gene.However, dsRNA having greater than 90%, or 95% sequence identity may beused in the present invention, and thus sequence variations that mightbe expected due to genetic mutation, strain polymorphism, orevolutionary divergence can be tolerated. The dsRNA is typically 100%complementary to the target RNA, but in some embodiments, the dsRNA maycontain single or multiple base-pair random mismatches between the RNAand the target gene.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a vesicle of lipids coating a reducedaqueous interior comprising a nucleic acid such as an iRNA agent or aplasmid from which an iRNA agent is transcribed. SNALPs are described,e.g., in U.S. Patent Application Publication Nos. 20060240093,20070135372, and U.S. Ser. No. 61/045,228 filed Apr. 15, 2008. Theseapplications are hereby incorporated by reference.

“Introducing into a cell,” when referring to a dsRNA, means facilitatinguptake or absorption into the cell, as is understood by those skilled inthe art. Absorption or uptake of dsRNA can occur through unaideddiffusive or active cellular processes, or by auxiliary agents ordevices. The meaning of this term is not limited to cells in vitro; adsRNA may also be “introduced into a cell,” wherein the cell is part ofa living organism. In such instance, introduction into the cell willinclude the delivery to the organism. For example, for in vivo delivery,dsRNA can be injected into a tissue site or administered systemically.In vitro introduction into a cell includes methods known in the art suchas electroporation and lipofection.

The terms “silence” and “inhibit the expression of,” in as far as theyrefer to the Factor VII gene, herein refer to the at least partialsuppression of the expression of the Factor VII gene, as manifested by areduction of the amount of mRNA transcribed from the Factor VII genewhich may be isolated from a first cell or group of cells in which theFactor VII gene is transcribed and which has or have been treated suchthat the expression of the Factor VII gene is inhibited, as compared toa second cell or group of cells substantially identical to the firstcell or group of cells but which has or have not been so treated(control cells). The degree of inhibition is usually expressed in termsof

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to Factor VII genetranscription, e.g. the amount of protein encoded by the Factor VII genewhich is secreted by a cell, or the number of cells displaying a certainphenotype, e.g apoptosis. In principle, Factor VII gene silencing may bedetermined in any cell expressing the target, either constitutively orby genomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given siRNA inhibitsthe expression of the Factor VII gene by a certain degree and thereforeis encompassed by the instant invention, the assays provided in theExamples below shall serve as such reference.

For example, in certain instances, expression of the Factor VII gene issuppressed by at least about 20%, 25%, 35%, 40% or 50% by administrationof the double-stranded oligonucleotide featured in the invention. Insome embodiments, the Factor VII gene is suppressed by at least about60%, 70%, or 80% by administration of the double-strandedoligonucleotide. In other embodiments, the Factor VII gene is suppressedby at least about 85%, 90%, or 95% by administration of thedouble-stranded oligonucleotide.

The terms “treat”, “treatment,” and the like, refer to relief from oralleviation of an disease or disorder, such as a viral hemorrhagicfever. In the context of the present invention insofar as it relates toany of the other conditions recited herein below (e.g., a FactorVII—mediated condition other than a thrombotic disorder), the terms“treat,” “treatment,” and the like mean to relieve or alleviate at leastone symptom associated with such condition, or to slow or reverse theprogression of such condition.

As used herein, the term “Factor VII-mediated condition or disease” andrelated terms and phrases refer to a condition or disorder characterizedby inappropriate, e.g., greater than normal, Factor VII activity.Inappropriate Factor VII functional activity might arise as the resultof Factor VII expression in cells which normally do not express FactorVII, or increased Factor VII expression (leading to, e.g., a symptom ofa viral hemorrhagic fever, or a thrombus). A Factor VII-mediatedcondition or disease may be completely or partially mediated byinappropriate Factor VII functional activity. However, a FactorVII-mediated condition or disease is one in which modulation of FactorVII results in some effect on the underlying condition or disorder(e.g., a Factor VII inhibitor results in some improvement in patientwell-being in at least some patients).

A “hemorrhagic fever” includes a combination of illnesses caused by aviral infection. Fever and gastrointestinal symptoms are typicallyfollowed by capillary hemorrhaging.

A “coagulopathy” is any defect in the blood clotting mechanism of asubject.

As used herein, a “thrombotic disorder” is any disorder characterized byunwanted blood coagulation.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management of aviral hemorrhagic fever, or an overt symptom of such disorder, e.g.,hemorraging, fever, weakness, muscle pain, headache, inflammation, orcirculatory shock. The specific amount that is therapeutically effectivecan be readily determined by ordinary medical practitioner, and may varydepending on factors known in the art, such as, e.g. the type ofthrombotic disorder, the patient's history and age, the stage of thedisease, and the administration of other agents.

As used herein, a “pharmaceutical composition” includes apharmacologically effective amount of a dsRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an RNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 25% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a25% reduction in that parameter.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract.

As used herein, a “transformed cell” is a cell into which a vector hasbeen introduced from which a dsRNA molecule may be expressed.

II. Double-Stranded Ribonucleic Acid (dsRNA)

In one embodiment, the invention provides double-stranded ribonucleicacid (dsRNA) molecules for inhibiting the expression of the Factor VIIgene in a cell or mammal. The dsRNA includes an antisense strandincluding a region of complementarity which is complementary to thecorresponding region of an mRNA formed in the expression of the FactorVII gene, and wherein the region of complementarity is less than 30nucleotides in length, generally 19-24 nucleotides in length. In oneembodiment the dsRNA, upon contact with a cell expressing said FactorVII gene, inhibits the expression of said Factor VII gene, e.g., in acell-based assay, by at least 25%, e.g., by at least 40%. The dsRNAincludes two RNA strands that are sufficiently complementary tohybridize to form a duplex structure. The sense strand includes a regionwhich is complementary to the antisense strand, such that the twostrands hybridize and form a duplex structure when combined undersuitable conditions. Generally, the duplex structure is between 15 and30, more generally between 18 and 25, yet more generally between 19 and24, and most generally between 21 and 23 base pairs in length.Similarly, the region of complementarity to the target sequence isbetween 15 and 30, more generally between 18 and 25, yet more generallybetween 19 and 24, and most generally between 21 and 23 nucleotides inlength. The dsRNA targeting FVII may further comprise one or moresingle-stranded nucleotide overhang(s). The dsRNA can be synthesized bystandard methods known in the art as further discussed below, e.g., byuse of an automated DNA synthesizer, such as are commercially availablefrom, for example, Biosearch, Applied Biosystems, Inc. In oneembodiment, the Factor VII gene is the human Factor VII gene. Inspecific embodiments, the first sequence is selected from the groupconsisting of the sense sequences of Tables 1, 2, and 3, and the secondsequence is selected from the group consisting of the antisensesequences of Tables 1, 2, and 3. In one embodiment, the cleavage iswithin 6, 5, 4, 3, 2 or 1 nucleotides of the cleavage site for a dsRNAfrom Tables 1, 2, and 3.

In further embodiments, the dsRNA includes at least one nucleotidesequence selected from the groups of sequences provided in Tables 1, 2,and 3. In other embodiments, the dsRNA includes at least two sequencesselected from this group, wherein one of the at least two sequences iscomplementary to another of the at least two sequences, and one of theat least two sequences is substantially complementary to a sequence ofan mRNA generated in the expression of the Factor VII gene. Generally,the dsRNA includes two oligonucleotides, wherein one oligonucleotide isdescribed as the sense strand in Tables 1, 2, or 3, and the secondoligonucleotide is described as the antisense strand in Tables 1, 2, or3.

The skilled person is well aware that dsRNAs including a duplexstructure of between 20 and 23, but specifically 21, base pairs havebeen identified as particularly effective in inducing RNA interference(Elbashir et al., EMBO 2001, 20:6877-6888). However, others have foundthat shorter or longer dsRNAs can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Tables 1, 2, and 3, the dsRNAsfeatured in the invention can comprise at least one strand of a lengthof minimally 21 nt. It can be reasonably expected that shorter dsRNAsincluding one of the sequences of Tables 1, 2 or 3 minus only a fewnucleotides on one or both ends may be similarly effective as comparedto the dsRNAs described above. Hence, dsRNAs including a partialsequence of at least 15, 16, 17, 18, 19, 20, or more contiguousnucleotides from one of the sequences of Tables 1, 2, or 3, anddiffering in their ability to inhibit the expression of the Factor VIIgene in a FACS assay as described herein below by not more than 5, 10,15, 20, 25, or 30% inhibition from a dsRNA including the full sequence,are contemplated by the invention.

In addition, the dsRNAs provided in Tables 1, 2, and 3 identify selectedsites in the Factor VII mRNA that are susceptible to RNAi basedcleavage. As such, the invention further includes dsRNAs that targetwithin the sequence targeted by one of the agents of the presentinvention. As used herein, a second dsRNA is said to target within thesequence of a first dsRNA if the second dsRNA cleaves the messageanywhere within the mRNA that is complementary to the antisense strandof the first dsRNA. Such a second agent will generally consist of atleast 15 contiguous nucleotides from one of the sequences provided inTables 1, 2, or 3 coupled to additional nucleotide sequences taken fromthe region contiguous to the selected sequence in the Factor VII gene.

The dsRNA featured in the invention can contain one or more mismatchesto the target sequence. In one embodiment, the dsRNA contains no morethan 3 mismatches. If the antisense strand of the dsRNA containsmismatches to a target sequence, the area of mismatch is typically notlocated in the center of the region of complementarity. If the antisensestrand of the dsRNA contains mismatches to the target sequence, then themismatch is typically restricted to 5 nucleotides from either end, forexample 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of theregion of complementarity. For example, for a 23 nucleotide dsRNA strandthat is complementary to a region of the Factor VII gene, the dsRNAgenerally does not contain any mismatch within the central 13nucleotides. The methods described herein can be used to determinewhether a dsRNA containing a mismatch to a target sequence is effectivein inhibiting the expression of the Factor VII gene. Consideration ofthe efficacy of dsRNAs with mismatches in inhibiting expression of theFactor VII gene is important, especially if the particular region ofcomplementarity in the Factor VII gene is known to have polymorphicsequence variation within the population.

In one embodiment, at least one end of the dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties than their blunt-ended counterparts.

Moreover, the present inventors have discovered that the presence ofonly one nucleotide overhang strengthens the interference activity ofthe dsRNA, without affecting its overall stability. dsRNA having onlyone overhang has proven particularly stable and effective in vivo, aswell as in a variety of cells, cell culture mediums, blood, and serum.Generally, the single-stranded overhang is located at the 3′-terminalend of the antisense strand or, alternatively, at the 3′-terminal end ofthe sense strand. The dsRNA may also have a blunt end, generally locatedat the 5′-end of the antisense strand. Such dsRNAs have improvedstability and inhibitory activity, thus allowing administration at lowdosages, i.e., less than 5 mg/kg body weight of the recipient per day.In one embodiment, the antisense strand of the dsRNA has 1-10 nucleotideoverhangs each at the 3′ end and the 5′ end over the sense strand. Inone embodiment, the sense strand of the dsRNA has 1-10 nucleotidesoverhangs each at the 3′ end and the 5′ end over the antisense strand.In another embodiment, one or more of the nucleotides in the overhang isreplaced with a nucleoside thiophosphate.

In yet another embodiment, the dsRNA is chemically modified to enhancestability. For example, the nucleic acids of the dsRNAs targeting FVIImay be synthesized and/or modified by methods well established in theart, such as those described in “Current protocols in nucleic acidchemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., NewYork, N.Y., USA, which is hereby incorporated herein by reference.Specific examples of dsRNA compounds include dsRNAs containing modifiedbackbones or no natural internucleoside linkages. As defined in thisspecification, dsRNAs having modified backbones include those thatretain a phosphorus atom in the backbone and those that do not have aphosphorus atom in the backbone. For the purposes of this specification,and as sometimes referenced in the art, modified dsRNAs that do not havea phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Typical modified dsRNA backbones include, for example,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; and 5,625,050, each of which is herein incorporated byreference

Typical modified dsRNA backbones that do not include a phosphorus atomtherein have backbones that are formed by short chain alkyl orcycloalkyl internucleoside linkages, mixed heteroatoms and alkyl orcycloalkyl internucleoside linkages, or ore or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other typical dsRNA mimetics, both the sugar and the internucleosidelinkage, i.e., the backbone, of the nucleotide units are replaced withnovel groups. The base units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligomeric compound,a dsRNA mimetic that has been shown to have excellent hybridizationproperties, is referred to as a peptide nucleic acid (PNA). In PNAcompounds, the sugar backbone of a dsRNA is replaced with an amidecontaining backbone, in particular an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. Representative U.S.patents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each ofwhich is herein incorporated by reference. Further teaching of PNAcompounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

In typical embodiments, dsRNAs have phosphorothioate backbones andoligonucleosides with heteroatom backbones, and in particular—CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) orMMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and—N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone isrepresented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No.5,489,677, and the amide backbones of the above-referenced U.S. Pat. No.5,602,240. In other embodiments, the dsRNAs featured in the inventionhave morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified dsRNAs may also contain one or more substituted sugar moieties.Typical dsRNAs include one of the following at the 2′ position: OH; F;O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; orO-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl may besubstituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl andalkynyl. Typical modifications include O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)nCH₃)]₂, where n and m are from 1 to about 10. Inother embodiments, dsRNAs include one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl,aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃,SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an dsRNA, or a group for improving thepharmacodynamic properties of an dsRNA, and other substituents havingsimilar properties. In one embodiment, the modification includes2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl)or 2′-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., analkoxy-alkoxy group. In other embodiments, modifications include2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as2′-DMAOE, as described in examples hereinbelow, and2′-dimethylaminoethoxyethoxy (also known in the art as2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions on the dsRNA, particularly the 3′ position ofthe sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs andthe 5′ position of 5′ terminal nucleotide. DsRNAs may also have sugarmimetics such as cyclobutyl moieties in place of the pentofuranosylsugar. Representative U.S. patents that teach the preparation of suchmodified sugar structures include, but are not limited to, U.S. Pat.Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;5,670,633; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

DsRNAs may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., Angewandte Chemie,International Edition, 1991, 30, 613, and those disclosed by Sanghvi, YS., Chapter 15, DsRNA Research and Applications, pages 289-302, Crooke,S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds. These include 5-substituted pyrimidines,6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T.and Lebleu, B., Eds., DsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and represent typical base substitutions,particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,30; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

Another modification of the dsRNAs targeting FVII involves chemicallinkage of the dsRNA to one or more moieties or conjugates that enhancethe activity, cellular distribution or cellular uptake of the dsRNA.Such moieties include but are not limited to lipid moieties such as acholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 199,86, 6553-6556), cholic acid (Manoharan et al., Biorg. Med. Chem. Let.,1994 4 1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan etal., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Biorg.Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser etal., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g.,dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991,10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethyl-ammonium1,2-di-O-hexadecyl-rac-glycero-3-Hphosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937).

Representative U.S. patents that teach the preparation of such dsRNAconjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979;4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538;5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045;5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263;4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136;5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506;5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552;5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696;5,599,923; 5,599,928 and 5,688,941, each of which is herein incorporatedby reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an dsRNA. The present invention also includesdsRNA compounds which are chimeric compounds. “Chimeric” dsRNA compoundsor “chimeras,” in the context of this invention, are dsRNA compounds,particularly dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a dsRNA compound. These dsRNAs typically contain at leastone region wherein the dsRNA is modified so as to confer upon the dsRNAincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the dsRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of dsRNA inhibition ofgene expression. Consequently, comparable results can often be obtainedwith shorter dsRNAs when chimeric dsRNAs are used, compared tophosphorothioate deoxydsRNAs hybridizing to the same target region.Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the dsRNA may be modified by a non-ligand group. Anumber of non-ligand molecules have been conjugated to dsRNAs in orderto enhance the activity, cellular distribution or cellular uptake of thedsRNA, and procedures for performing such conjugations are available inthe scientific literature. Such non-ligand moieties have included lipidmoieties, such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci.USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem.Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg.Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al.,Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiolor undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111;Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie,1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl.Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995,36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264:229), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923). Representative United States patents thatteach the preparation of such dsRNA conjugates have been listed above.Typical conjugation protocols involve the synthesis of dsRNAs bearing anaminolinker at one or more positions of the sequence. The amino group isthen reacted with the molecule being conjugated using appropriatecoupling or activating reagents. The conjugation reaction may beperformed either with the dsRNA still bound to the solid support orfollowing cleavage of the dsRNA in solution phase. Purification of thedsRNA conjugate by HPLC typically affords the pure conjugate. The use ofa cholesterol conjugate, for example, can increase targeting vaginalepithelium cells, a site of Factor VII expression expression.

Vector Encoded RNAi Agents

The dsRNAs targeting FVII can also be expressed from recombinant viralvectors intracellularly in vivo. For example, recombinant viral vectorscan include sequences encoding the dsRNA and any suitable promoter forexpressing the dsRNA sequences. Suitable promoters include, for example,the U6 or H1 RNA pol III promoter sequences and the cytomegaloviruspromoter. Selection of other suitable promoters is within the skill inthe art. The recombinant viral vectors can also include inducible orregulatable promoters for expression of the dsRNA in a particular tissueor in a particular intracellular environment. The use of recombinantviral vectors to deliver dsRNA to cells in vivo is discussed in moredetail below.

dsRNA targeting FVII can be expressed from a recombinant viral vectoreither as two separate, complementary RNA molecules, or as a single RNAmolecule with two complementary regions.

Any viral vector capable of accepting the coding sequences for the dsRNAmolecule(s) to be expressed can be used, for example vectors derivedfrom adenovirus (AV); adeno-associated virus (AAV); retroviruses (e.g,lentiviruses (LV), Rhabdoviruses, murine leukemia virus); herpes virus,and the like. The tropism of viral vectors can be modified bypseudotyping the vectors with envelope proteins or other surfaceantigens from other viruses, or by substituting different viral capsidproteins, as appropriate.

For example, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes. For example,an AAV vector expressing a serotype 2 capsid on a serotype 2 genome iscalled AAV 2/2. This serotype 2 capsid gene in the AAV 2/2 vector can bereplaced by a serotype 5 capsid gene to produce an AAV 2/5 vector.Techniques for constructing AAV vectors which express different capsidprotein serotypes are within the skill in the art; see, e.g., RabinowitzJ E et al. (2002), J Virol 76:791-801, the entire disclosure of which isherein incorporated by reference.

Selection of recombinant viral vectors suitable for use in theinvention, methods for inserting nucleic acid sequences for expressingthe dsRNA into the vector, and methods of delivering the viral vector tothe cells of interest are within the skill in the art. See, for example,Dornburg R (1995), Gene Therap. 2: 301-310; Eglitis M A (1988),Biotechniques 6: 608-614; Miller A D (1990), Hum Gene Therap. 1: 5-14;Anderson W F (1998), Nature 392: 25-30; and Rubinson D A et al., Nat.Genet. 33: 401-406, the entire disclosures of which are hereinincorporated by reference.

Typical viral vectors are those derived from AV and AAV. In oneembodiment, the dsRNA targeting FVII is expressed as two separate,complementary single-stranded RNA molecules from a recombinant AAVvector including, for example, either the U6 or H1 RNA promoters, or thecytomegalovirus (CMV) promoter.

A suitable AV vector for expressing a dsRNA targeting FVII, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Suitable AAV vectors for expressing the dsRNA targeting FVII, methodsfor constructing the recombinant AV vector, and methods for deliveringthe vectors into target cells are described in Samulski R et al. (1987),J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70:520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat.No. 5,252,479; U.S. Pat. No. 5,139,941; International Patent ApplicationNo. WO 94/13788; and International Patent Application No. WO 93/24641,the entire disclosures of which are herein incorporated by reference.

III. Pharmaceutical Compositions Including dsRNA

In one embodiment, the invention provides pharmaceutical compositionsincluding a dsRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition including the dsRNAis useful for treating a disease or disorder associated with theexpression or activity of the Factor VII gene, such as pathologicalprocesses mediated by Factor VII expression. Such pharmaceuticalcompositions are formulated based on the mode of delivery. One exampleis compositions that are formulated for systemic administration viaparenteral delivery.

The pharmaceutical compositions featured in the invention areadministered in dosages sufficient to inhibit expression of the FactorVII gene. The present inventors have found that, because of theirimproved efficiency, compositions including the dsRNAs targeting FVIIcan be administered at surprisingly low dosages. A maximum dosage of 5mg dsRNA per kilogram body weight (e.g., 1 mg/kg, 1.5 mg/kg, 2 mg/kg,2.5 mg/kg, 3 mg/kg, 3.5 mg/kg, 4 mg/kg, 4.5 mg/kg) of recipient per dayis sufficient to inhibit or completely suppress expression of the FactorVII gene.

In general, a suitable dose of dsRNA will be in the range of 0.01 to 5.0milligrams per kilogram body weight of the recipient per day, generallyin the range of 1 microgram to 1 mg per kilogram body weight per day.The pharmaceutical composition may be administered once daily, or thedsRNA may be administered as two, three, or more sub-doses atappropriate intervals throughout the day or even using continuousinfusion or delivery through a controlled release formulation. In thatcase, the dsRNA contained in each sub-dose must be correspondinglysmaller in order to achieve the total daily dosage. The dosage unit canalso be compounded for delivery over several days, e.g., using aconventional sustained release formulation which provides sustainedrelease of the dsRNA over a several day period. Sustained releaseformulations are well known in the art and are particularly useful forvaginal delivery of agents, such as could be used with the agents of thepresent invention. In this embodiment, the dosage unit contains acorresponding multiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual dsRNAs encompassed by theinvention can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by Factor VII expression. Such models are used for in vivotesting of dsRNA, as well as for determining a therapeutically effectivedose.

The present invention also includes pharmaceutical compositions andformulations which include the dsRNA compounds targeting FVII. Thepharmaceutical compositions may be administered in a number of waysdepending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration may be topical, pulmonary, e.g.,by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal), oralor parenteral. Admininstration may also be designed to result inpreferential localization to particular tissues through local delivery,e.g. by direct intraarticular injection into joints, by rectaladministration for direct delivery to the gut and intestines, byintravaginal administration for delivery to the cervix and vagina, byintravitreal administration for delivery to the eye. Parenteraladministration includes intravenous, intraarterial, intraarticular,subcutaneous, intraperitoneal or intramuscular injection or infusion; orintracranial, e.g., intrathecal or intraventricular, administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Topical formulations include those in which thedsRNAs are in admixture with a topical delivery agent such as lipids,liposomes, fatty acids, fatty acid esters, steroids, chelating agentsand surfactants. Typical lipids and liposomes include neutral (e.g.dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl cholineDMPC, distearolyphosphatidyl choline) negative (e.g.dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA). DsRNAs may be encapsulated within liposomes or mayform complexes thereto, in particular to cationic liposomes.Alternatively, dsRNAs may be complexed to lipids, in particular tocationic lipids. Typical fatty acids and esters include but are notlimited arachidonic acid, oleic acid, eicosanoic acid, lauric acid,caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid,linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein,dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, anacylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester (e.g.isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceuticallyacceptable salt thereof. Topical formulations are described in detail inU.S. patent application Ser. No. 09/315,298 filed on May 20, 1999 whichis incorporated herein by reference in its entirety.

In one embodiment, a FVII dsRNA featured in the invention is fullyencapsulated in the lipid formulation (e.g., to form a SPLP, pSPLP,SNALP, or other nucleic acid-lipid particle). As used herein, the term“SNALP” refers to a stable nucleic acid-lipid particle, including SPLP.As used herein, the term “SPLP” refers to a nucleic acid-lipid particlecomprising plasmid DNA encapsulated within a lipid vesicle. SNALPs andSPLPs typically contain a cationic lipid, a non-cationic lipid, and alipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). SNALPs and SPLPs are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). SPLPs include“pSPLP,” which include an encapsulated condensing agent-nucleic acidcomplex as set forth in PCT Publication No. WO 00/03683. The particlesof the present invention typically have a mean diameter of about 50 nmto about 150 nm, more typically about 60 nm to about 130 nm, moretypically about 70 nm to about 110 nm, most typically about 70 to about90 nm, and are substantially nontoxic. In addition, the nucleic acidswhen present in the nucleic acid-lipid particles of the presentinvention are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.C1),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.C1),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), or amixture thereof. The cationic lipid may comprise from about 20 mol % toabout 50 mol % or about 40 mol % of the total lipid present in theparticle.

The non-cationic lipid may be an anionic lipid or a neutral lipidincluding, but not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoyl-phosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid may be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles may be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HC1 (MW 1487) (Formula 1),Cholesterol (Sigma-Aldrich), and PEG-Ceramide C 16 (Avanti Polar Lipids)can be used to prepare lipid-siRNA nanoparticles (i.e., LNP01particles). Stock solutions of each in ethanol can be prepared asfollows: ND98, 133 mg/mL; Cholesterol, 25 mg/mL, PEG-Ceramide C16, 100mg/mL. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions canthen be combined in a, e.g., 42:48:10 molar ratio. The combined lipidsolution can be mixed with aqueous siRNA (e.g., in sodium acetate pH 5)such that the final ethanol concentration is about 35-45% and the finalsodium acetate concentration is about 100-300 mM. Lipid-siRNAnanoparticles typically form spontaneously upon mixing. Depending on thedesired particle size distribution, the resultant nanoparticle mixturecan be extruded through a polycarbonate membrane (e.g., 100 nm cut-off)using, for example, a thermobarrel extruder, such as Lipex Extruder(Northern Lipids, Inc). In some cases, the extrusion step can beomitted. Ethanol removal and simultaneous buffer exchange can beaccomplished by, for example, dialysis or tangential flow filtration.Buffer can be exchanged with, for example, phosphate buffered saline(PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1,about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano ZS(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totalsiRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated siRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total siRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” siRNA content (as measured by thesignal in the absence of surfactant) from the total siRNA content.Percent entrapped siRNA is typically >85%. For SNALP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Typical oral formulationsare those in which dsRNAs are administered in conjunction with one ormore penetration enhancers surfactants and chelators. Typicalsurfactants include fatty acids and/or esters or salts thereof, bileacids and/or salts thereof. Typical bile acids/salts includechenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA),cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid,glycholic acid, glycodeoxycholic acid, taurocholic acid,taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodiumglycodihydrofusidate. Typical fatty acids include arachidonic acid,undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid,myristic acid, palmitic acid, stearic acid, linoleic acid, linolenicacid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, anacylcholine, or a monoglyceride, a diglyceride or a pharmaceuticallyacceptable salt thereof (e.g. sodium). In some embodiments, formulationsinclude combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. In one embodiment, thecombination is the sodium salt of lauric acid, capric acid and UDCA.Further penetration enhancers include polyoxyethylene-9-lauryl ether,polyoxyethylene-20-cetyl ether. DsRNAs targeting FVII may be deliveredorally, in granular form including sprayed dried particles, or complexedto form micro or nanoparticles. DsRNA complexing agents includepoly-amino acids; polyimines; polyacrylates; polyalkylacrylates,polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins,starches, acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Typical complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyomithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S.application. Ser. No. 08/886,829 (filed Jul. 1, 1997), Ser. No.09/108,673 (filed Jul. 1, 1998), Ser. No. 09/256,515 (filed Feb. 23,1999), Ser. No. 09/082,624 (filed May 21, 1998) and Ser. No. 09/315,298(filed May 20, 1999), each of which is incorporated herein by referencein their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product. The preparation of such compositions andformulations is generally known to those skilled in the pharmaceuticaland formulation arts and may be applied to the formulation of thecompositions of the present invention.

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogenous systems of one liquiddispersed in another in the form of droplets usually exceeding 0.1.mu.min diameter (Idson, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p.245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemsincluding two immiscible liquid phases intimately mixed and dispersedwith each other. In general, emulsions may be of either the water-in-oil(w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finelydivided into and dispersed as minute droplets into a bulk oily phase,the resulting composition is called a water-in-oil (w/o) emulsion.Alternatively, when an oily phase is finely divided into and dispersedas minute droplets into a bulk aqueous phase, the resulting compositionis called an oil-in-water (o/w) emulsion. Emulsions may containadditional components in addition to the dispersed phases, and theactive drug which may be present as a solution in either the aqueousphase, oily phase or itself as a separate phase. Pharmaceuticalexcipients such as emulsifiers, stabilizers, dyes, and anti-oxidants mayalso be present in emulsions as needed. Pharmaceutical emulsions mayalso be multiple emulsions that are comprised of more than two phasessuch as, for example, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger andBanker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (Rieger, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic andcomprise a hydrophilic and a hydrophobic portion. The ratio of thehydrophilic to the hydrophobic nature of the surfactant has been termedthe hydrophile/lipophile balance (HLB) and is a valuable tool incategorizing and selecting surfactants in the preparation offormulations. Surfactants may be classified into different classes basedon the nature of the hydrophilic group: nonionic, anionic, cationic andamphoteric (Rieger, in Pharmaceutical Dosage Forms, Lieberman, Riegerand Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (Idson, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 199). Emulsion formulations for oral delivery have beenvery widely used because of ease of formulation, as well as efficacyfrom an absorption and bioavailability standpoint (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil baselaxatives, oil-soluble vitamins and high fat nutritive preparations areamong the materials that have commonly been administered orally as o/wemulsions.

In one embodiment of the present invention, the compositions of dsRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution(Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).Typically microemulsions are systems that are prepared by firstdispersing an oil in an aqueous surfactant solution and then adding asufficient amount of a fourth component, generally an intermediatechain-length alcohol to form a transparent system. Therefore,microemulsions have also been described as thermodynamically stable,isotropically clear dispersions of two immiscible liquids that arestabilized by interfacial films of surface-active molecules (Leung andShah, in: Controlled Release of Drugs: Polymers and Aggregate Systems,Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215).

Microemulsions commonly are prepared via a combination of three to fivecomponents that include oil, water, surfactant, cosurfactant andelectrolyte. Whether the microemulsion is of the water-in-oil (w/o) oran oil-in-water (o/w) type is dependent on the properties of the oil andsurfactant used and on the structure and geometric packing of the polarheads and hydrocarbon tails of the surfactant molecules (Schott, inRemington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa.,1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared toconventional emulsions, microemulsions offer the advantage ofsolubilizing water-insoluble drugs in a formulation of thermodynamicallystable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (Constantinideset al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm.Sci., 1996, 85, 138-143). Often microemulsions may form spontaneouslywhen their components are brought together at ambient temperature. Thismay be particularly advantageous when formulating thermolabile drugs,peptides or dsRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of dsRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofdsRNAs and nucleic acids within the gastrointestinal tract, vagina,buccal cavity and other areas of administration.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the dsRNAs and nucleicacids of the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Liposomes

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to cross intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis Liposomes fall into two broadclasses. Cationic liposomes are positively charged liposomes whichinteract with the negatively charged DNA molecules to form a stablecomplex.

The positively charged DNA/liposome complex binds to the negativelycharged cell surface and is internalized in an endosome. Due to theacidic pH within the endosome, the liposomes are ruptured, releasingtheir contents into the cell cytoplasm (Wang et al., Biochem. Biophys.Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g. as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemsincluding non-ionic surfactant and cholesterol. Non-ionic liposomalformulations including Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes including one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) includesone or more glycolipids, such as monosialoganglioside GM1, or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes including one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside GM1, galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes including (1)sphingomyelin and (2) the ganglioside GM1 or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomesincluding sphingomyelin. Liposomes including1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499 (Limet al.).

Many liposomes including lipids derivatized with one or more hydrophilicpolymers, and methods of preparation thereof, are known in the art.Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778) describedliposomes including a nonionic detergent, 2C1215G, that contains a PEGmoiety. Illum et al. (FEBS Lett., 1984, 167, 79) noted that hydrophiliccoating of polystyrene particles with polymeric glycols results insignificantly enhanced blood half-lives. Synthetic phospholipidsmodified by the attachment of carboxylic groups of polyalkylene glycols(e.g., PEG) are described by Sears (U.S. Pat. Nos. 4,426,330 and4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235) describedexperiments demonstrating that liposomes includingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes including a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes includingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al.).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A limited number of liposomes including nucleic acids are known in theart. WO 96/40062 to Thierry et al. discloses methods for encapsulatinghigh molecular weight nucleic acids in liposomes. U.S. Pat. No.5,264,221 to Tagawa et al. discloses protein-bonded liposomes andasserts that the contents of such liposomes may include a dsRNA RNA.U.S. Pat. No. 5,665,710 to Rahman et al. describes certain methods ofencapsulating oligodeoxynucleotides in liposomes. WO 97/04787 to Love etal. discloses liposomes including dsRNA dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g. they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N. Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N. Y., 1988, p. 285).

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly dsRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the abovementioned classes of penetration enhancers are described below ingreater detail.

Surfactants: In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of dsRNAs through the mucosa isenhanced. In addition to bile salts and fatty acids, these penetrationenhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (Leeet al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92); and perfluorochemical emulsions, such as FC-43. Takahashi et al.,J. Pharm. Pharmacol., 1988, 40, 252).

Fatty acids: Various fatty acids and their derivatives which act aspenetration enhancers include, for example, oleic acid, lauric acid,capric acid (n-decanoic acid), myristic acid, palmitic acid, stearicacid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₁₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (Lee et al.,Critical Reviews in Therapeutic Drug Carryier Systems, 1991, p.92;Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

Bile salts: The physiological role of bile includes the facilitation ofdispersion and absorption of lipids and fat-soluble vitamins (Brunton,Chapter 38 in: Goodman & Gilman's The Pharmacological Basis ofTherapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996,pp. 934-935). Various natural bile salts, and their syntheticderivatives, act as penetration enhancers. Thus the term “bile salts”includes any of the naturally occurring components of bile as well asany of their synthetic derivatives. Bile salts include, for example,cholic acid (or its pharmaceutically acceptable sodium salt, sodiumcholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid(sodium deoxycholate), glucholic acid (sodium glucholate), glycholicacid (sodium glycocholate), glycodeoxycholic acid (sodiumglycodeoxycholate), taurocholic acid (sodium taurocholate),taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid(sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodiumtauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate andpolyoxyethylene-9-lauryl ether (POE) (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents: Chelating agents, as used in connection with thepresent invention, can be defined as compounds that remove metallic ionsfrom solution by forming complexes therewith, with the result thatabsorption of dsRNAs through the mucosa is enhanced. With regards totheir use as penetration enhancers in the present invention, chelatingagents have the added advantage of also serving as DNase inhibitors, asmost characterized DNA nucleases require a divalent metal ion forcatalysis and are thus inhibited by chelating agents (Jarrett, J.Chromatogr., 1993, 618, 315-339). Chelating agents include but are notlimited to disodium ethylenediaminetetraacetate (EDTA), citric acid,salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines)(Lee et al., CriticalReviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi,Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33;Buur et al., J. Control Rel., 1990, 14, 43-51).

Non-chelating non-surfactants: As used herein, non-chelatingnon-surfactant penetration enhancing compounds can be defined ascompounds that demonstrate insignificant activity as chelating agents oras surfactants but that nonetheless enhance absorption of dsRNAs throughthe alimentary mucosa (Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33). This class of penetration enhancersinclude, for example, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Agents that enhance uptake of dsRNAs at the cellular level may also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipient suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Certain embodiments featured in the invention provide pharmaceuticalcompositions containing (a) one or more dsRNA molecules and (b) one ormore other therapeutic agents which function by a non-dsRNA-mediatedmechanism. For example, the one or more other therapeutic agents includeanticoagulants. Exemplary anticoagulants include, e.g., Warfarin(COUMADIN™); LMWH (Low Molecular Weight Heparins); factor Xa inhibitors,e.g, bisamidine compounds, and phenyl and naphthylsulfonamides;unfractionated heparin; aspirin; and platelet glycoprotein IIb/IIIablockers.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Suitable compounds typically exhibit high therapeutic indices.

The data obtained from cell culture assays and animal studies can beused in formulation a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method featured herein, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range of the compound or, when appropriate, of thepolypeptide product of a target sequence (e.g., achieving a decreasedconcentration of the polypeptide) that includes the IC50 (i.e., theconcentration of the test compound which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Such informationcan be used to more accurately determine useful doses in humans. Levelsin plasma may be measured, for example, by high performance liquidchromatography.

In addition to their administration individually or as a plurality, asdiscussed above, the dsRNAs targeting FVII can be administered incombination with other known agents effective in treatment ofpathological processes mediated by Factor VII expression. In any event,the administering physician can adjust the amount and timing of dsRNAadministration on the basis of results observed using standard measuresof efficacy known in the art or described herein.

Methods for Treating Diseases Caused by Expression of the Factor VIIGene

In one embodiment, the invention provides a method for treating asubject having a pathological condition mediated by the expression ofthe Factor VII gene, such as a viral hemorrhagic fever. In thisembodiment, the dsRNA acts as a therapeutic agent for controlling theexpression of the Factor VII protein. The method includes administeringa pharmaceutical composition to the patient (e.g., human), such as apatient infected with a virus, such that expression of the Factor VIIgene is silenced. Because of their high specificity, the dsRNAs featuredin the invention specifically target mRNAs of the Factor VII gene.

As used herein, the term “Factor VII-mediated condition or disease” andrelated terms and phrases refer to a condition or disorder characterizedby unwanted or inappropriate, e.g., abnormal Factor VII activity.Inappropriate Factor VII functional activity might arise as the resultof Factor VII expression in cells which normally do not express FactorVII, or increased Factor VII expression and/or activity (leading to,e.g., a symptom of a viral hemorrhagic fever, or a thrombotic disorder).A Factor VII-mediated condition or disease may be completely orpartially mediated by inappropriate Factor VII functional activity whichmay result by way of inappropriate activation of Factor VII. Regardless,a Factor VII-mediated condition or disease is one in which modulation ofFactor VII via RNA interference results in some effect on the underlyingcondition or disorder (e.g., a Factor VII inhibitor results in someimprovement in patient well-being in at least some patients).

The anti-Factor VII dsRNAs of the present invention may be used to treator diagnose a viral hemorrhagic fever in a subject. Treatment methodsinclude administering to a subject an anti-Factor VII dsRNA describeherein in an amount effective to treat the hemorrhagic fever.

Pathological processes refer to a category of biological processes thatproduce a deleterious effect. For example, unregulated expression ofFactor VII is associated with viral hemorrhagic fever, thromboticdisorders and cancer. A compound featured in the invention can typicallymodulate a pathological process when the compound reduces the degree orseverity of the process. For example, a hemorrhagic fever can beprevented, or related pathological processes can be modulated, by theadministration of a dsRNA that reduces or otherwise modulates theexpression of or at least one activity of Factor VII.

The dsRNA molecules featured herein may therefore also be used to treator prevent a viral hemorrhagic fever. The dsRNA can treat or prevent ahemorrhagic fever by ameliorating and/or preventing coagulopathy or aninflammatory response.

The dsRNA molecules featured herein may also be used to treat athrombotic disorder. Thrombotic disorders that can be treated with adsRNA that targets Factor VII include, but are not limited to, a localthrombus, acute myocardial infarction, unstable angina, an occlusivecoronary thrombus, or deep vein thrombosis.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraarticular, intraperitoneal, subcutaneous, intravitreal,transdermal, airway (aerosol), nasal, rectal, vaginal and topical(including buccal and sublingual) administration, and epiduraladministration. In some embodiments, the pharmaceutical compositions areadministered intraveneously by infusion or injection.

Methods for Inhibiting Expression of the Factor VII Gene

In yet another aspect, the invention provides a method for inhibitingthe expression of the Factor VII gene in a mammal. The method includesadministering a composition featured in the invention to the mammal suchthat expression of the target Factor VII gene is silenced. Because oftheir high specificity, the dsRNAs featured in the inventionspecifically target RNAs (primary or processed) of the target Factor VIIgene. Compositions and methods for inhibiting the expression of theFactor VII gene using dsRNAs can be performed as described elsewhereherein.

In one embodiment, the method includes administering a compositionincluding a dsRNA, wherein the dsRNA includes a nucleotide sequence thatis complementary to at least a part of an RNA transcript of the FactorVII gene of the mammal to be treated. When the organism to be treated isa mammal such as a human, the composition may be administered by anymeans known in the art including, but not limited to oral or parenteralroutes, including intravenous, intramuscular, intraarticular,intracranial, subcutaneous, intravitreal, transdermal, airway (aerosol),nasal, rectal, vaginal and topical (including buccal and sublingual)administration. In certain embodiments, the compositions areadministered by intraveneous infusion or injection.

dsRNA Expression Vectors

In another aspect, FVII specific dsRNA molecules that modulate FVII geneexpression activity are expressed from transcription units inserted intoDNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10;Skillern, A., et al., International PCT Publication No. WO 00/22113,Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S.Pat. No. 6,054,299). These transgenes can be introduced as a linearconstruct, a circular plasmid, or a viral vector, which can beincorporated and inherited as a transgene integrated into the hostgenome. The transgene can also be constructed to permit it to beinherited as an extrachromosomal plasmid (Gassmann, et al., Proc. Natl.Acad. Sci. USA (1995) 92:1292).

The individual strands of a dsRNA can be transcribed by promoters on twoseparate expression vectors and co-transfected into a target cell.Alternatively each individual strand of the dsRNA can be transcribed bypromoters both of which are located on the same expression plasmid. Inone embodiment, a dsRNA is expressed as an inverted repeat joined by alinker polynucleotide sequence such that the dsRNA has a stem and loopstructure.

The recombinant dsRNA expression vectors are generally DNA plasmids orviral vectors. dsRNA expressing viral vectors can be constructed basedon, but not limited to, adeno-associated virus (for a review, seeMuzyczka, et al., Curr. Topics Micro. Immunol. (1992) 158:97-129));adenovirus (see, for example, Berkner, et al., BioTechniques (1998)6:616), Rosenfeld et al. (1991, Science 252:431-434), and Rosenfeld etal. (1992), Cell 68:143-155)); or alphavirus as well as others known inthe art. Retroviruses have been used to introduce a variety of genesinto many different cell types, including epithelial cells, in vitroand/or in vivo (see, e.g., Eglitis, et al., Science (1985)230:1395-1398; Danos and Mulligan, Proc. Natl. Acad. Sci. USA (1998)85:6460-6464; Wilson et al., 1988, Proc. Natl. Acad. Sci. USA85:3014-3018; Armentano et al., 1990, Proc. Natl. Acad. Sci. USA87:61416145; Huber et al., 1991, Proc. Natl. Acad. Sci. USA88:8039-8043; Ferry et al., 1991, Proc. Natl. Acad. Sci. USA88:8377-8381; Chowdhury et al., 1991, Science 254:1802-1805; vanBeusechem. et al., 1992, Proc. Nad. Acad. Sci. USA 89:7640-19; Kay etal., 1992, Human Gene Therapy 3:641-647; Dai et al., 1992, Proc.Natl.Acad. Sci. USA 89:10892-10895; Hwu et al., 1993, J. Immunol.150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. No. 4,980,286; PCTApplication WO 89/07136; PCT Application WO 89/02468; PCT Application WO89/05345; and PCT Application WO 92/07573). Recombinant retroviralvectors capable of transducing and expressing genes inserted into thegenome of a cell can be produced by transfecting the recombinantretroviral genome into suitable packaging cell lines such as PA317 andPsi-CRIP (Comette et al., 1991, Human Gene Therapy 2:5-10; Cone et al.,1984, Proc. Natl. Acad. Sci. USA 81:6349). Recombinant adenoviralvectors can be used to infect a wide variety of cells and tissues insusceptible hosts (e.g., rat, hamster, dog, and chimpanzee) (Hsu et al.,1992, J. Infectious Disease, 166:769), and also have the advantage ofnot requiring mitotically active cells for infection.

The promoter driving dsRNA expression in either a DNA plasmid or viralvector may be a eukaryotic RNA polymerase I (e.g. ribosomal RNApromoter), RNA polymerase II (e.g. CMV early promoter or actin promoteror U1 snRNA promoter) or generally RNA polymerase III promoter (e.g. U6snRNA or 7SK RNA promoter) or a prokaryotic promoter, for example the T7promoter, provided the expression plasmid also encodes T7 RNA polymeraserequired for transcription from a T7 promoter. The promoter can alsodirect transgene expression to the pancreas (see, e.g. the insulinregulatory sequence for pancreas (Bucchini et al., 1986, Proc. Natl.Acad. Sci. USA 83:2511-2515)).

In addition, expression of the transgene can be precisely regulated, forexample, by using an inducible regulatory sequence and expressionsystems such as a regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of transgene expression in cells or inmammals include regulation by ecdysone, by estrogen, progesterone,tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (EPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the dsRNA transgene.

Generally, recombinant vectors capable of expressing dsRNA molecules aredelivered as described below, and persist in target cells.Alternatively, viral vectors can be used that provide for transientexpression of dsRNA molecules. Such vectors can be repeatedlyadministered as necessary. Once expressed, the dsRNAs bind to target RNAand modulate its function or expression. Delivery of dsRNA expressingvectors can be systemic, such as by intravenous or intramuscularadministration, by administration to target cells ex-planted from thepatient followed by reintroduction into the patient, or by any othermeans that allows for introduction into a desired target cell.

dsRNA expression DNA plasmids are typically transfected into targetcells as a complex with cationic lipid carriers (e.g. Oligofectamine) ornon-cationic lipid-based carriers (e.g. Transit-TKO™). Multiple lipidtransfections for dsRNA-mediated knockdowns targeting different regionsof a single Factor VII gene or multiple Factor VII genes over a periodof a week or more are also contemplated by the invention. Successfulintroduction of the vectors into host cells can be monitored usingvarious known methods. For example, transient transfection can besignaled with a reporter, such as a fluorescent marker, such as GreenFluorescent Protein (GFP). Stable transfection of ex vivo cells can beensured using markers that provide the transfected cell with resistanceto specific environmental factors (e.g., antibiotics and drugs), such ashygromycin B resistance.

The Factor VII specific dsRNA molecules can also be inserted intovectors and used as gene therapy vectors for human patients. Genetherapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (see U.S. Pat. No.5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In case of conflict, the present specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

Examples Example 1 Design of siRNA to target FVII

siRNA design was performed to identify siRNAs targeting coagulationfactor VII (also known as AI132620, Cf7, Coagulation factor VIIprecursor, coagulation factor VII, FVII, Serum prothrombin conversionaccelerator, FVII coagulation protein, and eptacog alfa).

Human mRNA sequences to two FVII transcript variants, RefSeq ID number:NM_(—)000131.3 (3141 bp) (see, e.g., FIGS. 7A and 7B) and NM_(—)019616.2(3075 bp) (GenBank record dated Nov. 18, 2007) (see, e.g., FIGS. 8A and8B), were used. Rhesus sequences were assembled from NCBI and ENSEMBLdatabase sources (see below). siRNA duplexes cross-reactive to human andrhesus monkey (Macaca mulatta) FVII with predicted specificity to humanFVII were designed. Twenty-four duplexes were synthesized for screening,and these are shown in Table 1.

Human-rhesus cross-reactivity. Human-rhesus cross-reactivity was definedas prerequisite for in silico selection of siRNAs. For this, it wasassured that two curated human variants for FVII and available rhesussequences contained 19mer siRNA target sites.

Human mRNA sequences to two FVII transcript variants were downloadedfrom NCBI Nucleotide database; one of these, NM_(—)000131.3, was furtherused as the reference sequence.

Sequences for rhesus FVII mRNAs downloaded from the NCBI Nucleotidedatabase (NM_(—)001080136.1-2424 bp and D21212.1-478 bp, partial cds),and ENSEMBL database (ENSMMUT00000001477-1389 bp andENSMMUT00000042997-1326 bp) were aligned to build a consensus sequencefor rhesus monkey FVII with a total length of 2424 bp.

All possible 19mers were extracted from the human mRNA referencesequence, resulting in a pool of candidate target sites corresponding to3122 (sense strand) sequences of NM 000131.3-reactive FVII siRNAs.

To determine siRNAs reactive towards both curated human variants and theconsensus rhesus sequences, each candidate siRNA target site wassearched in the human RefSeq sequence NM_(—)019616.2 and the partialrhesus sequence. The resulting siRNAs were defined as human-rhesuscross-reactive siRNAs.

Specificity Prediction.

The predicted specificity of the siRNA was used as criterion for finalselection, manifested by targeting human FVII mRNA sequences, but notother human mRNAs.

To identify human FVII-specific siRNAs that will avoid targetingnon-FVII human transcripts (potential “off-target” genes), human-rhesuscross-reactive siRNAs were subjected to a homology search against thehuman RefSeq mRNA database which was considered to represent thecomprehensive human transcriptome.

For this, the fastA algorithm was used to determine the most homologoushit region for antisense and sense strands to each sequence of the humanRefSeq database (release 24).

Resulting alignments with every RefSeq entry were further analyzed by aperl script to extract the number and position of mismatches, and basedon this, to calculate a specificity score. siRNA strands were assigned acategory of specificity according to the calculated specificity scores:a score above three qualified as highly specific, equal to three asspecific, and between 2.2 and 2.8 as moderate specific.

siRNA Sequence Selection.

For selection of siRNAs, a specificity score of 2.8 or more for theantisense strand, and 2 or more for the sense strand, was chosen as aprerequisite for selection of siRNAs, whereas all sequences containingfour or more consecutive G's (poly-G sequences) were excluded.

Twenty-four siRNA sequences, cross-reactive to all human and rhesusmonkey FVII mRNAs mentioned above, and passing the specificitycriterion, were selected (see Table 1). The resulting set of twenty-fourconsists of two highly specific, 16 specific, and six moderatelyspecific siRNAs (considering specificity of the antisense strand only).

Example 2 Silencing of FVII In Vivo

Rats (n=4) were given a single i.v. injection of siFVII formulated withthe lipidoid formulation 98N12-5 at doses of 1.25, 2.5, 3, 3.5, 4, 5,and 10 mg/kg.

Animals were bled and sacrificed 48 h after administration. Significant,dose-dependent reductions in liver Factor VII mRNA levels were observed,with 40%, 80%, and greater than 90% silencing at 1.25, 2.5, and 5 mg/kg,respectively (FIG. 1). No silencing was observed using a formulatedcontrol siRNA (siCont), demonstrating specificity of silencing. Thereduction in liver Factor VII mRNA levels produced a concomitantdose-dependent reduction in serum Factor VII protein levels, with nearlycomplete silencing at the highest dose levels (FIG. 2).

As would be expected, significantly reduced serum Factor VII levelsproduced a phenotypic effect in the treated animals. As Factor VII ispart of the extrinsic coagulation pathway, treated animals had impairedclotting through this pathway as measured by prolongation in prothrombintime (PT) (FIG. 3). The phenotypic effect was found to be specific andnot attributable to the delivery vehicle, as the formulated controlgroup demonstrated no perturbations in PT. The resultant gene silencingwas highly durable. Single injections of 98N12-5 formulated FactorVII-targeting siRNA (siFVII) were capable of mediating silencingpersisting for nearly 4 weeks.

The sequences for the sense and antisense strands of the siRNAs are asfollows.

siFVII: sense: SEQ ID NO: 1 5′-GGAucAucucAAGucuuAcT*T-3′ antisense:SEQ ID NO: 2 5′-GuAAGAcuuGAGAuGAuccT*T-3′ siCont: sense: SEQ ID NO: 35′-cuuAcGcuGAGuAcuucGAT*T-3′ antisense: SEQ ID NO: 45′-UCGAAGuACUcAGCGuAAGT*T-3′

2′-O-Me modified nucleotides are in lower case, 2′-Fluoro modifiednucleotides are in bold lower case, and phosphorothioate linkages arerepresented by asterisks. siRNAs were generated by annealing equimolaramounts of complementary sense and antisense strands.

All animal procedures used were approved by the Institutional AnimalCare and Use Committee (IACUC) and were consistent with local, state,and federal regulations as applicable. C57BL/6 mice (Charles River Labs,MA) and Sprague-Dawley rats (Charles River Labs, MA) received eithersaline or siRNA in lipidoid formulations via tail vein injection at avolume of 0.01 mL/g. Serum levels of Factor VII protein were determinedin samples collected by retroorbital bleed using a chromogenic assay(Coaset Factor VII, DiaPharma Group, OH or Biophen FVII, AniaraCorporation, OH). Liver mRNA levels of Factor VII were determined usinga branched DNA assay (QuantiGene Assay, Panomics, Calif.).

Lipidoid-based siRNA formulations included lipidoid, cholesterol,poly(ethylene glycol)-lipid (PEG-lipid), and siRNA. Formulations wereprepared using a protocol similar to that described by Semple andcolleagues (Maurer et al. Biophys. J. 80:2310-2326, 2001; Semple et al.,Biochim. Biophys. Acta 1510:152-166, 2001). Stock solutions of98N12-5(1).4HC1 MW 1489, mPEG2000-Ceramide C16 (Avanti Polar Lipids) MW2634 or mPEG2000-DMG MW 2660, and cholesterol MW 387 (Sigma-Aldrich)were prepared in ethanol and mixed to yield a molar ratio of 42:10:48.Mixed lipids were added to 125 mM sodium acetate buffer pH 5.2 to yielda solution containing 35% ethanol, resulting in spontaneous formation ofempty lipidoid nanoparticles. Resulting nanoparticles were extrudedthrough a 0.08 membrane (2 passes). siRNA in 35% ethanol and 50 mMsodium acetate pH 5.2 was added to the nanoparticles at 1:7.5 (wt:wt)siRNA:total lipids and incubated at 37° C. for 30 min. Ethanol removaland buffer exchange of siRNA-containing lipidoid nanoparticles wasachieved by tangential flow filtration against phosphate buffered salineusing a 100,000 MWCO membrane. Finally, the formulation was filteredthrough a 0.2μ sterile filter. Particle size was determined using aMalvem Zetasizer NanoZS (Malvem, UK). siRNA content was determined by UVabsorption at 260 nm and siRNA entrapment efficiency was determined byRibogreen assay 32. Resulting particles had a mean particle diameter ofapproximately 50 nm, with peak width of 20 nm, and siRNA entrapmentefficiency of >95%. See also PCT/US2007/080331.

TABLE 4 Abbreviations of nucleoside monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleoside(s) A adenosine Ccytidine G guanosine U uridine N any nucleotide (G, A, C, U, or dT) a2′-O-methyladenosine c 2′-O-methylcytidine g 2′-O-methylguanosine u2′-O-methyluridine dT 2′-deoxythymidine s a phosphorothioate linkage

Example 3 Factor VII as a Target for the Treatment of Viral HemorrhagicFevers

Robust in vivo silencing of Factor VII in hepatocytes in mice wasobserved following administration of a lipid-formulated FVII dsRNA(LNP-01 FVII) (FIG. 4). Mice were treated intravenously with the dsRNA,and serum was analyzed for FVII protein 24 hrs later. The decrease inFVII protein levels occurred in a dose-dependent manner. An LNP-01formulated luciferase siRNA was used as a negative control.

Preliminary data using a non-optimized liposomally-formulated dsRNA toFactor VII showed a beneficial survival effect in mice infected withEbola virus (FIG. 5). Mice were treated with LNP-01 formulated siRNA atday 0 (5 mg/kg i.v.) and at day 3 (3 mg/kg i.p.) after infection with30,000 LD50 of Ebola-Zaire. Mice were monitored for survival with n=10per treatment group. Negative controls included untreated mice and micetreated with LNP-01 formulated luciferase siRNA. The observed result wasconsistent with the benefit seen with recombinant anti-coagulant NapC2(Geisbert et al., 2003, Lancet, 362:1953-1958) and was particularlyencouraging given the reduced role for coagulopathy in the mouse modelversus that seen in Ebola-infected non-human primates and humans.

Example 4 Treatment with siFVII Exhibited an Extended Effect

C57BL/6 mice were treated with a single bolus i.v. injection ofLNP01-siFVII at 5 mg/kg. FIG. 6 shows that this administration causeddecreased FVII levels that lasted for more than 3 weeks.

Example 5 dsRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

siRNA Synthesis

Single-stranded RNAs were produced by solid phase synthesis on a scaleof 1 μmole using an Expedite 8909 synthesizer (Applied Biosystems,Applera Deutschland GmbH, Darmstadt, Germany) and controlled pore glass(CPG, 500 Å, Proligo Biochemie GmbH, Hamburg, Germany) as solid support.RNA and RNA containing 2′-O-methyl nucleotides were generated by solidphase synthesis employing the corresponding phosphoramidites and2′-O-methyl phosphoramidites, respectively (Proligo Biochemie GmbH,Hamburg, Germany). These building blocks were incorporated at selectedsites within the sequence of the oligoribonucleotide chain usingstandard nucleoside phosphoramidite chemistry such as described inCurrent protocols in nucleic acid chemistry, Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA. Phosphorothioatelinkages were introduced by replacement of the iodine oxidizer solutionwith a solution of the Beaucage reagent (Chruachem Ltd, Glasgow, UK) inacetonitrile (1%). Further ancillary reagents were obtained fromMallinckrodt Baker (Griesheim, Germany).

Deprotection and purification of the crude oligoribonucleotides by anionexchange HPLC were carried out according to established procedures.Yields and concentrations were determined by UV absorption of a solutionof the respective RNA at a wavelength of 260 nm using a spectralphotometer (DU 640B, Beckman Coulter GmbH, UnterschleiBheim, Germany).Double stranded RNA was generated by mixing an equimolar solution ofcomplementary strands in annealing buffer (20 mM sodium phosphate, pH6.8; 100 mM sodium chloride), heated in a water bath at 85-90° C. for 3minutes and cooled to room temperature over a period of 3-4 hours. Theannealed RNA solution was stored at −20° C. until use.

For the synthesis of 3′-cholesterol-conjugated siRNAs (herein referredto as -Chol-3′), an appropriately modified solid support is used for RNAsynthesis. The modified solid support is prepared as follows:

Diethyl-2-azabutane-1,4-dicarboxylate AA

A 4.7 M aqueous solution of sodium hydroxide (50 mL) is added into astirred, ice-cooled solution of ethyl glycinate hydrochloride (32.19 g,0.23 mole) in water (50 mL). Then, ethyl acrylate (23.1 g, 0.23 mole) isadded and the mixture is stirred at room temperature until completion ofthe reaction is ascertained by TLC. After 19 h the solution ispartitioned with dichloromethane (3×100 mL). The organic layer is driedwith anhydrous sodium sulfate, filtered and evaporated. The residue isdistilled to afford AA (28.8 g, 61%).

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonyl-amino)-hexanoyl]-amino}-propionicacid ethyl ester AB

Fmoc-6-amino-hexanoic acid (9.12 g, 25.83 mmol) is dissolved indichloromethane (50 mL) and cooled with ice. Diisopropylcarbodiimde(3.25 g, 3.99 mL, 25.83 mmol) is added to the solution at 0° C. It isthen followed by the addition of Diethyl-azabutane-1,4-dicarboxylate (5g, 24.6 mmol) and dimethylamino pyridine (0.305 g, 2.5 mmol). Thesolution is brought to room temperature and stirred further for 6 h.Completion of the reaction is ascertained by TLC. The reaction mixtureis concentrated under vacuum and ethyl acetate is added to precipitatediisopropyl urea. The suspension is filtered. The filtrate is ished with5% aqueous hydrochloric acid, 5% sodium bicarbonate and water. Thecombined organic layer is dried over sodium sulfate and concentrated togive the crude product which is purified by column chromatography (50%EtOAC/Hexanes) to yield 11.87 g (88%) of AB.

3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC

3-{Ethoxycarbonylmethyl-[6-(9H-fluoren-9-ylmethoxycarbonylamino)-hexanoyl]-amino}-propionicacid ethyl ester AB (11.5 g, 21.3 mmol) is dissolved in 20% piperidinein dimethylformamide at 0° C. The solution is continued stirring for 1h. The reaction mixture is concentrated under vacuum, water is added tothe residue, and the product is extracted with ethyl acetate. The crudeproduct is purified by conversion into its hydrochloride salt.

3-({6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}ethoxycarbonylmethyl-amino)-propionicacid ethyl ester AD

The hydrochloride salt of3-[(6-Amino-hexanoyl)-ethoxycarbonylmethyl-amino]-propionic acid ethylester AC (4.7 g, 14.8 mmol) is taken up in dichloromethane. Thesuspension is cooled to 0° C. on ice. To the suspensiondiisopropylethylamine (3.87 g, 5.2 mL, 30 mmol) is added. To theresulting solution cholesteryl chloroformate (6.675 g, 14.8 mmol) isadded. The reaction mixture is stirred overnight. The reaction mixtureis diluted with dichloromethane and ished with 10% hydrochloric acid.The product is purified by flash chromatography (10.3 g, 92%).

1-{6-[17-(1,5-Dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-4-oxo-pyrrolidine-3-carboxylicacid ethyl ester AE

Potassium t-butoxide (1.1 g, 9.8 mmol) is slurried in 30 mL of drytoluene. The mixture is cooled to 0° C. on ice and 5 g (6.6 mmol) ofdiester AD is added slowly with stirring within 20 mins. The temperatureis kept below 5° C. during the addition. The stirring is continued for30 mins at 0° C. and 1 mL of glacial acetic acid is added, immediatelyfollowed by 4 g of NaH₂PO₄.H₂O in 40 mL of water. The resultant mixtureis extracted twice with 100 mL of dichloromethane each and the combinedorganic extracts are ished twice with 10 mL of phosphate buffer each,dried, and evaporated to dryness. The residue is dissolved in 60 mL oftoluene, cooled to 0° C. and extracted with three 50 mL portions of coldpH 9.5 carbonate buffer. The aqueous extracts are adjusted to pH 3 withphosphoric acid, and extracted with five 40 mL portions of chloroformwhich are combined, dried and evaporated to dryness. The residue ispurified by column chromatography using 25% ethylacetate/hexane toafford 1.9 g of b-ketoester (39%).

[6-(3-Hydroxy-4-hydroxymethyl-pyrrolidin-1-yl)-6-oxo-hexyl]-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AF

Methanol (2 mL) is added dropwise over a period of 1 h to a refluxingmixture of b-ketoester AE (1.5 g, 2.2 mmol) and sodium borohydride(0.226 g, 6 mmol) in tetrahydrofuran (10 mL). Stirring is continued atreflux temperature for 1 h. After cooling to room temperature, 1 N HCl(12.5 mL) is added, the mixture is extracted with ethylacetate (3×40mL). The combined ethylacetate layer is dried over anhydrous sodiumsulfate and concentrated under vacuum to yield the product which ispurified by column chromatography (10% MeOH/CHCl₃) (89%).

(6-{3-[Bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-4-hydroxy-pyrrolidin-1-yl}-6-oxo-hexyl)-carbamicacid17-(1,5-dimethyl-hexyl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-ylester AG

Diol AF (1.25 μm 1.994 mmol) is dried by evaporating with pyridine (2×5mL) in vacuo. Anhydrous pyridine (10 mL) and4,4′-dimethoxytritylchloride (0.724 g, 2.13 mmol) are added withstirring. The reaction is carried out at room temperature overnight. Thereaction is quenched by the addition of methanol. The reaction mixtureis concentrated under vacuum and to the residue dichloromethane (50 mL)is added. The organic layer is ished with 1M aqueous sodium bicarbonate.The organic layer is dried over anhydrous sodium sulfate, filtered andconcentrated. The residual pyridine is removed by evaporating withtoluene. The crude product is purified by column chromatography (2%MeOH/Chloroform, Rf=0.5 in 5% MeOH/CHCl₃) (1.75 g, 95%).

Succinic acidmono-(4-[bis-(4-methoxy-phenyl)-phenyl-methoxymethyl]-1-{6-[17-(1,5-dimethyl-hexyl)-10,13-dimethyl2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1Hcyclopenta[a]phenanthren-3-yloxycarbonylamino]-hexanoyl}-pyrrolidin-3-yl)ester AH

Compound AG (1.0 g, 1.05 mmol) is mixed with succinic anhydride (0.150g, 1.5 mmol) and DMAP (0.073 g, 0.6 mmol) and dried in a vacuum at 40°C. overnight. The mixture is dissolved in anhydrous dichloroethane (3mL), triethylamine (0.318 g, 0.440 mL, 3.15 mmol) is added and thesolution is stirred at room temperature under argon atmosphere for 16 h.It is then diluted with dichloromethane (40 mL) and ished with ice coldaqueous citric acid (5 wt %, 30 mL) and water (2×20 mL). The organicphase is dried over anhydrous sodium sulfate and concentrated todryness. The residue is used as such for the next step.

Cholesterol derivatised CPG AI

Succinate AH (0.254 g, 0.242 mmol) is dissolved in a mixture ofdichloromethane/acetonitrile (3:2, 3 mL). To that solution DMAP (0.0296g, 0.242 mmol) in acetonitrile (1.25 mL),2,2′-Dithio-bis(5-nitropyridine) (0.075 g, 0.242 mmol) inacetonitrile/dichloroethane (3:1, 1.25 mL) are added successively. Tothe resulting solution triphenylphosphine (0.064 g, 0.242 mmol) inacetonitrile (0.6 ml) is added. The reaction mixture turned brightorange in color. The solution is agitated briefly using a wrist-actionshaker (5 mins). Long chain alkyl amine-CPG (LCAA-CPG) (1.5 g, 61 mM) isadded. The suspension is agitated for 2 h. The CPG is filtered through asintered funnel and ished with acetonitrile, dichloromethane and ethersuccessively. Unreacted amino groups are masked using aceticanhydride/pyridine. The achieved loading of the CPG is measured bytaking UV measurement (37 mM/g).

The synthesis of siRNAs bearing a 5′-12-dodecanoic acid bisdecylamidegroup (herein referred to as “5′-C32-”) or a 5′-cholesteryl derivativegroup (herein referred to as “5′-Chol-”) is performed as described in WO2004/065601, except that, for the cholesteryl derivative, the oxidationstep is performed using the Beaucage reagent in order to introduce aphosphorothioate linkage at the 5′-end of the nucleic acid oligomer.

We claim:
 1. A double-stranded ribonucleic acid (dsRNA), wherein saiddsRNA comprises at least two sequences that are substantiallycomplementary to each other and wherein a sense strand of the dsRNAcomprises a first sequence and an antisense strand of the dsRNAcomprises a second sequence comprising a region that is substantiallycomplementary to at least part of an mRNA encoding Factor VII, whereineach strand is at least 15 nucleotides in length and less than or equalto 30 nucleotides in length, and wherein said first sequence is selectedfrom the group consisting of said sense strand sequences in Tables 1, 2,and 3, and wherein said second sequence is selected from the groupconsisting of said antisense strand sequences in Tables 1, 2, and
 3. 2.The dsRNA of claim 1, wherein the sense strand sequence comprises thesequence of SEQ ID NO:5, and the antisense strand sequence comprises thesequence of SEQ ID NO:6.
 3. The dsRNA of claim 1, wherein the dsRNA canreduce liver Factor VII mRNA levels in rats by at least 25% silencingwith a single administration of a dose 98N12-5 formulated FactorVII-targeting siRNA.
 4. The dsRNA of claim 1, wherein said dsRNAcomprises at least one modified nucleotide.
 5. The dsRNA of claim 4,wherein said modified nucleotide is chosen from the group consisting of:a 2′-O-methyl modified nucleotide, a nucleotide comprising a5′-phosphorothioate group, and a terminal nucleotide linked to acholesteryl derivative or dodecanoic acid bisdecylamide group.
 6. ThedsRNA of claim 4, wherein said modified nucleotide is chosen from thegroup consisting of: a 2′-deoxy-2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide,2′-amino-modified nucleotide, 2′-alkyl-modified nucleotide, morpholinonucleotide, a phosphoramidate, and a non-natural base comprisingnucleotide.
 7. The dsRNA of claim 1 comprising a phosphorothioate or a2′-modified nucleotide.
 8. The dsRNA of claim 1, wherein the region ofcomplementarity is at least 15 nucleotides in length.
 9. The dsRNA ofclaim 1, wherein the region of complementarity is 19-21 nucleotides inlength.
 10. A cell comprising the dsRNA of claim
 1. 11. A pharmaceuticalcomposition, comprising a dsRNA of claim 1 and a pharmaceuticallyacceptable carrier.
 12. A method for inhibiting the expression of aFactor VII gene in a cell, the method comprising: (a) introducing intothe cell a double-stranded ribonucleic acid (dsRNA) of claim 1; and (b)maintaining the cell produced in step (a) for a time sufficient toobtain degradation of the mRNA transcript of the Factor VII gene,thereby inhibiting expression of the Factor VII gene in the cell.
 13. Amethod of treating, preventing or managing a viral hemorrhagic fevercomprising administering to a patient in need of such treatment,prevention or management a therapeutically or prophylactically effectiveamount of a dsRNA of claim
 1. 14. A vector comprising a regulatorysequence operably linked to a nucleotide sequence that encodes at leastone strand of a dsRNA of claim
 1. 15. A cell comprising the vector ofclaim 14.